Polarity of extracts and fractions of four Combretum (Combretaceae) species used to treat infections and gastrointestinal disorders in southern African traditional medicine has a major effect on different relevant in vitro activities

Abstract

Ethnopharmacological importance: Gastrointestinal disorders and infections are the major pathoaetiologies of diarrhoea causing many problems in human health and animal production. Many Combretum species are used in traditional medicine to treat infectious diseases including diarrhoea and many other ailments by rural people in Africa and Asia. Much of the work done to date on this genus was on the non-polar or intermediate polarity components. Some parameters that may cause diarrhoea and the evaluation of more polar extracts have apparently not been investigated.

Aims: The polar components were extracted and fractionated by solvent-solvent fractionation to yield fractions with different polarities. The activity of these fractions on different parameters that could be involved in factors associated with diarrhoea was investigated. The cytotoxic activities of the extracts were also determined to evaluate the potential of these extracts to combat diarrhoea in production animals.

Materials and methods: Phenolic-enriched leaf extracts of Combretum bracteosum (COB), Combretum padoides (COP), Combretum vendae (COV) and Combretum woodii (COW) were obtained by extracting with a mixture of 70% acetone acidified with 1% HCl and n-hexane. Acetone was removed from a portion of the 70% acetone extract and it was sequentially treated by solvent-solvent fractionation with dichloromethane, ethyl acetate, and butanol to yield fractions with a large variation in polarity. The phenolic constituents of the extracts and fractions were determined using standard procedures The antioxidant activities were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2'-azino-bis (3-ethylbenzothiazoline)-6-sulphonic acid (ABTS(+)) radical scavenging, ferric reducing antioxidant power (FRAP) methods and lipid peroxidation inhibitory capacity standard methods. The ferric reducing antioxidant activities of the fractions were also determined. The minimum inhibitory concentrations (MICs) of the crude extracts and fractions against four bacterial and three fungal strains were assessed with a microplate serial dilution method. Cyclooxygenase (COX) and lipoxygenase (LOX) enzyme inhibitory assays and cytotoxicity studies against Vero cells were also carried out.

Result: Some of the fractions had much higher antioxidant activity than the positive controls. The average EC50 values of the extracts for the DPPH and ABTS antioxidant assays were 0.21-12µg/ml (COP), 0.25-16µg/ml (COV), 0.33-9.41µg/ml (COW) and 4.97-85µg/ml (COB) respectively while the mean EC50 values for the positive controls ascorbic acid and trolox were 1.28-1.51 and 1.02-1.19µg/ml respectively. All the crude extracts inhibited lipid peroxidation of linoleic acid by more than 80% at a concentration of 64 µg/ml. COP had the highest antibacterial activity with MICs ranging between 19-2500µg/ml, followed by COV with MICs ranging between 39-625µg/ml; COW and COB had similar MICs ranging between 39-2500µg/ml. COP also had the highest antifungal activity with MICs between 19-625µg/ml. The MIC for COW and COV ranged from 19 to 1250 µg/ml. COB had the lowest antifungal activity (MIC values were between 39 and 625 µg/ml). In general non-polar fractions had a high antimicrobial activity and polar fractions had a high antioxidant activity. The extracts had no activity against COX 1 and 2 enzymes in the anti-inflammatory assay but had good lipoxygenase inhibition. The crude extracts had high concentration of hydrolysable tannin (gallotannin). A good correlation (R(2)= 0.99) was found between the antioxidant activity and total tannin content indicating that, gallotannins may be responsible for the antioxidant activity.

Conclusion: The results obtained in this study with more polar extracts indicate that the use of extracts of these plant species as antidiarrhoeal agents may have a scientific basis. The extractant used here extracted a much higher percentage of the phytochemicals than acetone. It was better for isolating antioxidant compounds (polar) but not good for isolating antimicrobial compounds (non-polar) from the same species compared to acetone, ethyl acetate, dichloromethane, and hexane.

Keywords: Combretum; Cytotoxicity; Diarrhoea; Gastrointestinal disorders; Infection; Intestinal inflammation.